DETAILED ACTION
EXAMINER'S AMENDMENT
An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
Authorization for this examiner’s amendment was given in an interview with Michele Washington and Amber Majnik on 8/31/2026.
The application has been amended as follows:
Claim 1f) is changed from “repeating steps d) through f)” to “repeating steps c) through e)”.
Status of the Claims
Claims 1-8, 15-16, 18, 31, 47-50, 52-53, 75, and 105-106 are pending and examined herein.
Claims 9-14, 17, 19-30, 32-46, 51, 54-74, and 76-104 are canceled.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Objections
Claim 50 is objected to because of the following informalities: it recites the limitation of claim 1 further comprising a step "l)" Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 5-7, 47, 75 and 106 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1, 105, and 106 recite sets of barcoding primers comprising a list of features. However, the formatting and numbering of the lists renders the required features indefinite because it conflicts with the wording of the claim. For example, the wording of claim 1a indicates that the first set of barcoding primers comprises a ULS sequence, a first well-specific barcode, and a targeting region. On the other hand, the formatting and numbering of the list would imply that the first set of barcoding primers comprises (i) a ULS sequence, (ii) a first well-specific barcode, (iii) random hexamer sequences, and (iv) specific sequences. Similarly, claim 105 states that “the kit” comprises a first plurality of barcoding primers and an isothermal polymerase. However, the recited isothermal polymerase is not numbered, so it is unclear which group of components it belongs to. These issues are also present in claims 1d, 105a, 106b, and 106c. To overcome this rejection, it is suggested that the lists be re-formatted and numbered to accurately reflect the features required, perhaps making use of another level of indentation, where useful.
Claim 2 is dependent on claim 1 and recites the limitation “the target region”. There is insufficient antecedent basis for this limitation in the claim because neither claim 1 nor 2 recites “a target region”. Claim 1a does recite “a targeting region” and “target sequences”, but it is unclear which of these “the target region” refers to. For the purposes of examination, claim 2 will be interpreted as limiting “the targeting region” of claim 1a.
Claims 5-7 are dependent on claim 1 and recite the limitation "the barcoding primers". There is insufficient antecedent basis for this limitation in the claims because claim 1 recites several sets of barcoding primers (1a, 1d, 1f) which all comprise different features. It is unclear which set, if not all, of the barcoding primers are limited by claims 5-7. For the purposes of examination, claims 5 and 6 will be interpreted as limiting the first set of barcoding primers, as these are recited as comprising a targeting region.
Claim 48 recites the limitation “the terminal primer sequence”. There is insufficient antecedent basis for this limitation in claims 1 and 47, upon which claim 48 depends.
Claims 75 and 106 contain lists of features that do not have a conjunction such as “and” or “or”. Therefore, the claims are indefinite as it is unclear whether one or all of the features are required in order for the claim to be met. For the purposes of examination, the claims will be interpreted as using “and” and therefore requiring all elements to be present.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 5-6 and 53 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 5 and 6 are dependent on claim 1 and recite the limitation that the barcoding primers comprise only random hexamer sequences or specific sequences. This language indicates that the only sequences present in the barcoding primers are the random hexamer or specific sequences. However, claim 1 states that sets of barcoding primers comprise universal linker strand sequences and well-specific barcodes. Therefore, claims 5 and 6 do not include all the limitations of claim 1. This rejection may be overcome by limiting “the targeting region” as opposed to “the barcoding primer”, which is how these claims will be interpreted for the purposes of examination. Other solutions are possible.
Claim 53 is dependent on claim 1 and recites the limitation “wherein the ULS sequence is different in each round”. However, the second set of barcoding primers of claim 1d is recited as comprising “(vii) the ULS sequence…”, which finds antecedent basis in (i) of claim 1a. The invocation of antecedent basis indicates that the ULS sequence of the second set of barcoding primers is the same as the ULS sequence of the first set of barcoding primers. Therefore, the limitation of claim 53 for these sequences to be different fails to incorporate all limitations of claim 1. This rejection may be overcome by changing the second set of barcoding primers to not invoke antecedent basis with regards to the ULS sequence, for example: “(vii) a ULS sequence…”, which is how this claim will be interpreted for the purposes of examination. Other solutions are possible.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim 75 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Rosenberg et al. (Science, 2018, 360: 176-182; cited in IDS of 05/06/2025). Rosenberg discloses a method comprising: capturing barcoded amplicons (cDNA) comprising an affinity moiety (p. S3, Preparing Oligonucleotides…: “a biotin molecule”) by contacting the amplicons with an affinity capture reagent (p. S4, Purification of cDNA: streptavidin beads); converting the barcoded amplicons into double-stranded captured amplicons (p. S4-5, Template Switch); and amplifying the double-stranded captured amplicons to generate free amplification products that are not attached to the affinity moiety and affinity capture reagent (p. S5, PCR).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Steemers et al., Wells et al., and Kuchina et al.
Claims 1-4, 6, 47-49, and 53 are rejected under 35 U.S.C. 103 as being unpatentable over Steemers et al. (PG Pub No: US 2019/0382753; cited in IDS of 05/06/2025) in view of Wells et al. (PG Pub No: US 2023/0265499; effectively filed 8/14/2020) and Kuchina et al. (Science, 2021, 371: eaba5237; cited in IDS of 05/06/2025).
Regarding claims 1, 6, and 47, Steemers discloses a method comprising:
dividing a plurality of fixed and permeabilized cells into a plurality of wells (p. 12, ¶[0092]), each well comprising a first set of barcoding primers (p. 14, ¶[0108]) comprising: a universal linker strand (ULS) sequence (p. 14, ¶[0110]: “universal sequence”), a first well-specific barcode (p. 14, ¶[0108]: “index”), and a targeting region (p. 15, [0114]: complementary sequences);
amplifying genomic DNA while it remains inside of each cell to create barcoded molecules under conditions that maintain cellular membrane integrity (p. 19, ¶[0158]);
pooling the cells from the plurality of wells (p. 12, ¶[0092]: “after indexing, the subsets can be pooled);
dividing the cells into a plurality of wells (p. 12, ¶[0092]), each well comprising a second set of barcoding primers (p. 14, ¶[0108]) comprising: an adapter sequence complementary to the ULS sequence (p. 14, ¶[0110]: “in a subsequent step to anneal a nucleotide sequence that can be used as a primer for addition of another nucleotide sequence”), a second well-specific barcode (p. 14, ¶[0108]: “another index”), and a ULS sequence (p. 14, ¶[0110]: “another universal sequence”);
amplifying the barcoded molecules under conditions that maintain cellular membrane integrity (p. 19, ¶[0158]); and
repeating steps c) through e) for a plurality of rounds, wherein the well-specific barcode is different in each round (p. 15, ¶[0115]), and wherein the final round, the set of barcoding primers further comprises an affinity moiety to generate barcoded amplicons comprising the affinity moiety (p. 16, ¶[0123]: universal capture sequence).
Steemers further discloses lysis (p. 17, ¶[0139]), capture (p. 17, ¶[0141]), and amplification of the captured barcoded amplicons (p. 17-8, ¶[0142-3]). However, in contrast with the instant claim, Steemers discloses lysis prior to the final round of indexing. With regard to the order of steps, the courts have ruled that the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946; In re Gibson 39 F.2d 975, 5 USPZ 230 (CCPA 1930; MPEP 2144.04 section IV C). In the instant case, the steps of indexing and lysis are taught by Steemers and thus is prima facie obvious in the absence of new or unexpected results.
Steemers does not explicitly disclose amplification of genomic DNA while it remains in the cell to create barcoded molecules under conditions that maintain cellular membrane integrity. Wells discloses a method of in situ amplification (abstract) of gDNA (p.3, ¶[0038]; Figure 9) for the purpose of appending well-specific barcodes (p. 3, ¶[0040]) to a subset of fixed and permeabilized cells (p. 4, ¶[0043]). This method serves the same purpose as the method of adding well-specific barcodes of Steemers. Therefore, it would have been obvious to one of ordinary skill in the art by the effective filing date to substitute the method of adding well-specific barcodes from Wells for that of Steemers, yielding a split-pool barcoding method via in situ amplification. Furthermore, the result of this substitution would have been predictable as it utilizes much of the same techniques disclosed in Steemers. In fact, the disclosure of Steemers does generically recite the use of amplification for adding barcodes (p. 15, ¶[0114]).
Wells further discloses that the primers comprise specific sequences (p. 20-21, ¶[0220-1]).
Steemers also does not disclose amplifying the barcoded amplicons off of the affinity moiety and affinity capture reagent to generate free amplification products.
Kuchina discloses a method for split pool barcoding to generate barcoded amplicons where the amplicons are captured by an affinity moiety and affinity capture reagent that the amplicons are amplified off of to generate free amplification products (Supplemental p. 2, PCR: “on-beads PCR followed by the qPCR to amplify the product”). Because this step and the corresponding one of Steemers share the same function (to amplify the barcoded products), it would have been obvious to one of ordinary skill in the art to substitute the amplification of Steemers for the amplification off of affinity capture moieties of Kuchina. The results of this substitution would have been predictable, as amplification methods and nucleic acid immobilization chemistry are well-understood in the art.
Regarding claim 2, Wells discloses specific sequences as discussed above. Additionally, Steemers further discloses ligation of a dsDNA sequence comprising a terminal primer sequence (p. 15, ¶[0121]) prior to affinity capture.
Regarding claim 3, Steemers further discloses amplifying the barcoded amplicons with primers comprising specific sequences (p. 19, ¶[0155]).
Regarding claim 4, Steemers further discloses amplifying the barcoded amplicons with primers that comprise random sequences and a terminal primer sequence (p. 20, ¶[0159]). Steemers does not disclose random hexamers, but Steemers elsewhere discloses random priming sequences of 4 (p. 41, ¶[0338]: SEQ ID NO: 5) and 7 nucleotides (p. 41, ¶[0340]: SEQ ID NO: 6). MPEP 2144.05(I) states that ranges or amounts that "lie inside ranges disclosed by the prior art" are considered prima facie obvious. As a length of 6 bases lies within the range of random priming sequences disclosed by Steemers, random hexamers would have been obvious to one of ordinary skill in the art by the effective filing date.
Regarding claims 48 and 49, Steemers further discloses amplifying the barcoded amplicons with primers comprising specific sequences or universal sequences (p. 19, ¶[0155]).
Regarding claim 53, Steemers further discloses that the ULS sequence is different in each round, and wherein the adapter in each round is complementary to the ULS sequence of the previous round (p. 14, ¶[0110]).
Steemers et al., Wells et al., Kuchina et al., and Rosenberg et al.
Claims 50 and 52 are rejected under 35 U.S.C. 103 as being unpatentable over Steemers et al. in view of Wells et al. and Kuchina et al., as evidenced by Rosenberg et al. (Science, 2018, 360: 176-182; cited in IDS of 05/06/2025).
Regarding claims 50 and 52, Kuchina further discloses purifying and sequencing the amplification products (Supplemental p. 2, PCR: “performed according to the SPLiT-seq protocol”), as evidenced by Rosenberg (Supplemental p.5, Tagmentation: “PCR reaction was removed and purified… to generate an Illumina-compatible sequencing library”).
Steemers et al., Wells et al., Kuchina et al., and Gao et al.
Claim 5, 7-8, 15-16, and 105-106 are rejected under 35 U.S.C. 103 as being unpatentable over Steemers et al. in view of Wells et al. and Kuchina et al., and further in view of Gao et al. (TrAC (2019), 121: 115700).
Regarding claim 5, neither Steemers nor Wells disclose that the barcoding primers comprise random hexamer sequence and not specific sequences. However, Steemers does disclose that MDA (multiple displacement amplification) is a method of random primer amplification of genomic DNA (p. 19-20, ¶[0158]). Additionally, Steemers states that the first round of barcoding can be carried out using amplification via random priming (p. 13, ¶[0098]). Based on this suggestion, it would have been obvious to one of ordinary skill in the art by the effective filing date to apply multiple displacement amplification to the in situ amplification steps of the method of Steemers, Wells, and Kuchina, resulting in a method wherein the first set of barcoding primers comprise random sequences. As discussed in relation to claim 4, random hexamers are also obvious over the disclosure of Steemers. Additionally, there would have been a reasonable expectation of success as other methods of isothermal amplification have been successfully used in situ, such as rolling circle amplification, as evidenced by Gao (abstract).
Regarding claim 7, there is no necessary structural difference between a pool of primers containing random sequences and a pool of primers containing random AND specific sequences, particularly when both are recited to hybridize to sequences of gDNA, as a set of random sequences can also comprise specific sequences. Therefore, claim 7 is also rendered obvious by the disclosures of Steemers, Wells, Kuchina, and Gao, as above.
Regarding claims 8 and 15-16, Steemers further discloses that MDA comprises an isothermal amplification reaction that utilizes the isothermal polymerase phi29 (p. 19-20, ¶[0158]).
Regarding claims 105 and 106, the method of claim 5 would necessarily result in the combination of reagents as disclosed in the instant claims. Therefore, they are also rendered obvious.
Steemers et al., Wells et al., Kuchina et al., and Sasaki et al.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Steemers et al. in view of Wells et al. and Kuchina et al., and further in view of Sasaki et al. (Biotechnol. J. (2006), 1: 440-446). The limitations of claim 1 are rendered obvious by the disclosures of Steemers, Wells, and Kuchina, as discussed previously. Claim 18 recites contacting the fixed and permeabilized cells with a crowding agent during the amplification of genomic DNA within the cells, which is not explicitly disclosed in any of the previously applied prior art.
Sasaki discloses that conditions of molecular crowding, achieved by addition of polyethylene glycol, increases the efficiency of PCR (abstract). Therefore, it would have been obvious to one of ordinary skill in the art by the effective filing date to contact the fixed and permeabilized cells with a crowding agent based on the motivation to increase the efficiency of the amplification. There would have been a reasonable expectation of success as Sasaki teaches that this effect common across DNA polymerases, and therefore would be expected to achieve the same effect in different types of amplification reactions.
Steemers et al., Wells et al., Kuchina et al., and Compton
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Steemers et al. in view of Wells et al. and Kuchina et al. as applied to claims 1 and 3 above, and further in view of Compton (Nature (1991), 350: 91-92). Neither Steemers, Wells, nor Kuchina disclose amplification of barcoded amplicons using specific sequences and an isothermal amplification reaction. Compton teaches that nucleic acid sequence-based amplification (NASBA) is a recognized equivalent method for PCR (p. 91, col. 1, 1st and 2nd ¶) that comprises an isothermal amplification reaction (p. 91, col. 1, 2nd ¶: “NASBA is a continuous, isothermal process”). As this equivalency is known in the art, it would have been obvious to one of ordinary skill to substitute PCR with specific sequence primers for NASBA with specific sequence primers by the effective filing date.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Application No: 18/719,471
Claims 1-8, 15-16, 18, 31, 47-50, 52-53, 75, and 105-106 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-53 of copending Application No. 18719471 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because the reference claims disclose a method of split-pool labeling gDNA in fixed and permeabilized cells via in situ amplification with random and/or specific primers, amplification off of beads, isothermal amplification with phi29 polymerase, and crowding agents.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Application No: 19/553,141
Claims 1-8, 15-16, 18, 31, 47-50, 52-53, 75, and 105-106 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, and 5 of copending Application No. 19553141 in view of Steemers, Kuchina, Rosenberg, Gao, Sasaki, and Compton. The reference application discloses in situ isothermal amplification of gDNA from single cells, similar to the disclosure of Wells. Therefore, with the disclosures of all other prior art of record, the claims of the instant application are rendered obvious.
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims are allowed.
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/ALEXANDRA OLSON/Examiner, Art Unit 1684
/JEREMY C FLINDERS/Primary Examiner, Art Unit 1684
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